Hypothalamic inhibitory factor (HIF), an isomer of ouabain, is a physiologic inhibitor of membrane-bound Na+, K+-activated adenosine triphosphate (Na+, K+-ATPase) and produces a positive inotropic effect on cardiac cells. Positive inotropic effect means that the contractility of the cardiac cells is enhanced in a dose-dependent manner. HIF can be administered to a mammalian host to treat cardiac malfunction (e.g., congestive heart failure, paroxysmal atrial tachycardia, atrial fibrillation and flutter). Use of HIF for treating cardiac malfunction is further described in U.S. patent application Ser. No. 08/338,264, filed Nov. 10, 1994.
HIF has been purified to homogeneity using an affinity chromatography method in which purified canine renal Na+, K+-ATPase is coupled to paramagnetic particles through a glutaraldehyde bridge (Tymiak, A. A., et al. Proc. Natl. Acad. Sci., USA, 90:8189–8193 (1993)). The enzyme immobilizes bound HIF in the presence of Mg++ and inorganic phosphorous with high affinity, and after washing away contaminating materials, HIF is eluted from the affinity column by chelating Mg++ with EDTA. With a subsequent HPLC step, this technique produced enough pure HIF to allow structure determination and the differentiation of HIF from ouabain (Tymiak, A. A., et al. Proc. Natl. Acad. Sci., USA, 90:8189–8193 (1993)). Unfortunately, losses at the affinity step are substantial, with activity recovery averaging only 30%. Also, the method is laborious in the sense that pure enzyme needs to be repeatedly prepared from fresh canine renal medulla.
Thus, a need exists for an improved method of isolating and/or purifying HIF. In particular, there is a need for large-scale preparative methods for purifying HIF.